Beta-galactosidase Mutant, and Preparation Method and Application Thereof

ABSTRACT

The present invention discloses a β-galactosidase mutant, and a preparation method and application thereof, belonging to the fields of gene engineering and enzyme engineering. Amino acids of specific sites in the β-galactosidase are mutated, the β-galactosidase is transferred into a recombinant bacterium, and enzymatic transformation is performed under optimized conditions, so that the yield of galactooligosaccharide produced by the mutant reaches 59.8%, which is increased by about 70% as compared with that of wild enzyme, thereby implementing the increase of the galactooligosaccharide yield. The present invention has very high industrialized application value.

TECHNICAL FIELD

The present invention relates to a β-galactosidase mutant, and apreparation method and application thereof, belonging to the fields ofgene engineering and enzyme engineering.

BACKGROUND

Galactooligosaccharide (GOS) is a functional oligosaccharide that iscurrently widely used in the food industry. As a new functional foodadditive, the GOS has attracted worldwide attention because of itsunique physiological functions and excellent physicochemical properties.With the continuous advancing of development and research of GOS,coupled with abundant raw materials in China and the unlimited potentialof the consumer market, the production of GOS has already set off astrong momentum all over the country. At present, the production ofoligosaccharides in China is still an emerging industry. The developmentof GOS has not yet reached its scale. The main reason for restrictingthe production of GOS in China is the lack of industrial enzymes withexcellent performance. Therefore, it is very important to findindustrial enzymes with excellent performance.

β-galactosidase is a main enzyme used in industrial enzymatic methodproduction of galactooligosaccharide. β-galactosidase derived fromdifferent microorganisms has different ability to generate GOS due todifferent properties. Currently known better GOS-producing strains areB. circulans, Kluyveromyces Lactis and A. oryzae. Although the yield ofB. circulans (Bacillus circulans) is higher (48.3%), its product of GOSproduction is mainly 4′GalLac which has a poor probiotic effect. A mainproduct of GOS produced by A. oryzae as a food-safe strain is 6′GalLac.6′GalLac has a better probiotic effect than 4′GalLac, but its yield isonly about 19%, which greatly limits its application.

Therefore, improving the yield of Aspergillus oryzae derivedβ-galactosidase for producing GOS to create conditions for itsindustrial production is a technical problem to be solved at present.

SUMMARY

The present invention firstly aims to provide a β-galactosidase mutant,wherein the mutant results from mutation of one or more amino acid sitesof β-galactosidase of which an amino acid sequence is as shown in SEQ IDNO.2.

In an implementation of the present invention, the mutant results frommutation of one or more sites at the 140th, 264th, 304th and 806thpositions of the β-galactosidase of which an amino acid sequence is asshown in SEQ ID NO.2.

In an implementation of the present invention, the mutant results frommutation of asparagine (Asn) at the 140th position of theβ-galactosidase of which the amino acid sequence is as shown in SEQ IDNO.2 to cysteine (Cys), wherein the mutant is named N140C.

In an implementation of the present invention, the mutant results frommutation of phenylalanine (Phe) at the 264th position of theβ-galactosidase of which the amino acid sequence is as shown in SEQ IDNO.2 to tryptophan (Trp), wherein the mutant is named F264W.

In an implementation of the present invention, the mutant results frommutation of phenylalanine (Phe) at the 304th position of theβ-galactosidase of which the amino acid sequence is as shown in SEQ IDNO.2 to glutamine (Gln), wherein the mutant is named F304Q.

In an implementation of the present invention, the mutant results frommutation of tryptophan (Trp) at the 806th position of theβ-galactosidase of which the amino acid sequence is as shown in SEQ IDNO.2 to phenylalanine (Phe), wherein the mutant is named W806F.

In an implementation of the present invention, the mutant results frommutation of phenylalanine (Phe) at the 264th position of theβ-galactosidase of which the amino acid sequence is as shown in SEQ IDNO.2 to tryptophan (Trp) and asparagine (Asn) at the 140th position tocysteine (Cys), wherein the mutant is named N140C/F264W.

In an implementation of the present invention, the mutant results frommutation of asparagine (Asn) at the 140th position of theβ-galactosidase of which the amino acid sequence is as shown in SEQ IDNO.2 to cysteine (Cys) and tryptophan (Trp) at the 806th position tophenylalanine (Phe), wherein the mutant is named N140C/W806F.

The present invention secondly aims to provide a preparation method ofthe β-galactosidase mutant, which specifically comprises the followingsteps:

(1) according to determined mutant sites, designing mutagenic primers ofsite-directed mutagenesis, and performing site-directed mutagenesis byusing a vector carrying the β-galactosidase gene as a template; andconstructing a plasmid vector containing the gene coding the mutant;

(2) transforming a mutant plasmid into a host cell; and

(3) selecting a positive clone, performing fermentation culture, andperforming centrifuging, wherein supernate is a crude enzyme solution ofthe β-galactosidase mutant.

In an implementation of the present invention, the plasmid vector is anyof pET series or pPIC9k.

The present invention thirdly aims to provide a method for preparinggalactooligosaccharide by using the β-galactosidase mutant, whichspecifically comprises the following steps:

(1) by using lactose with concentration of 400 g/L-600 g/L as asubstrate, adding the β-galactosidase, and adding an acetic acid-sodiumacetate buffer, wherein pH is 4.5-5;

(2) performing enzymatic transformation in a shaking water bath, whereinreaction temperature is 40-60° C.;

(3) performing reaction within 5-36 h, wherein a sample is taken every 4hours during the reaction; and

(4) performing liquid-phase analysis on a reaction product, andcalculating yield.

In an implementation of the present invention, an addition amount of theβ-galactosidase is 2.5-5 U/mL.

The present invention has the following beneficial effects: byconstructing the Aspergillus oryzae derived β-galactosidase mutant, themaximum GOS yield by transforming lactose is increased from 35.2% of awild bacterium to 59.8%, so that the problem of lower industrialproduction yield of this enzyme is solved. Compared with the wild type,this enzyme has higher glucoside transformation activity, and issuitable for industrial production.

When the present invention is applied to galactooligosaccharide (GOS)production, optimum pH for production is 4.5, and optimum temperature is40° C., which is more suitable for industrial production.

Based on the constructed mutant, the present invention optimizes theproduction of galactooligosaccharide (GOS) by enzymatic transformation,so that the industrial production value is higher. By performingenzymatic transformation under optimum conditions, the yield reaches59.8%, which is increased by about 70% as compared with the wild type.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is optimum temperature for producing galactooligosaccharide bytransforming lactose by using a wild enzyme and a mutant;

FIG. 2 is optimum pH for producing galactooligosaccharide bytransforming lactose by using the wild type and the mutant.

DETAILED DESCRIPTION

BMGY liquid culture medium: YNB: 13.4 g/L; yeast extract: 10 g/L;peptone: 20 g/L; glycerol: 10 g/L; K₂HPO₄: 2.29 g/L; KH₂PO₄: 11.8 g/L;(NH₄)₂SO₄: 10 g/L;

BMMY liquid culture medium: YNB: 13.4g/L; yeast extract: 10g/L; peptone:20g/L; methanol: 40 g/L; K₂HPO₄: 2.29 g/L; KH₂PO₄: 11.8 g/L; (NH₄)₂SO₄:10 g/L.

Enzyme Activity Measuring Method:

β-galactosidase activity analysis useso-nitrophenyl-β-D-galactopyranoside (oNPG) as a substrate.

Prepare 20 mmol/L oNPG, take 100 μL of a properly diluted fermentationsolution or enzyme solution and 1800 μL of 0.1 mol/L pH4.5 sodiumacetate buffer, hold at the temperature of 60° C. for 5 min, adding 100μL of the substrate, hold at the temperature of 60° C. for 10 min,immediately adding 1 mL of 1 mol/L precooled Na2CO3 solution to stopreaction and perform color development, and measure absorbance at 420 nmwith a spectrophotometer.

Definition of enzyme activity unit: under the above analysis conditions,the amount of enzyme that catalytically produces 1 μmol of oNP perminute is one activity unit.

Analysis of Galactooligosaccharide (GOS) Yield by HPLC Method

A 60% (W/V) lactose solution is prepared as a substrate, and certainamounts of wild enzyme and double-mutant enzyme solutions are added, sothat the final enzyme activity of the wild type enzyme solution is 10U/mL and the final enzyme activity of the mutant is 2.5 U/mL (the effectof the amount of enzyme added on the production of GOS was previouslyperformed, and the above conditions were respectively the optimumamounts of enzyme added for the mutant and the wild type). The reactionis performed under the optimum temperature and pH conditions, whereinthe reaction for the wild type is performed for 10 h (the time of themaximum yield of the wild type in a pre-experiment), and the reactionfor the mutant is performed for 5 h (the time of the maximum yield ofthe mutant in the pre-experiment). The reaction solution is centrifugedat 12000 rpm for 10 min, and supernate is taken, filtered through a 0.22nm ultrafiltration membrane to remove 20 uL and subjected to HPLCanalysis.

In the reaction solution, the amounts of disaccharides, trisaccharides,tetrasaccharides and pentasaccharides in a product are analyzed by HPLC.Chromatographic conditions for measurement are: Agilent 1200 HPLCchromatograph, Agilent automatic sampler, chromatographic column Hi-PlexNa, (300*7.7mm), and differential detector Agilent G1362A; a mobilephase is pure water, a flow rate is 0.3 mL/min, and column temperatureis set at 80° C.

Chromatographic conditions for measuring disaccharides are: Agilent 1200HPLC chromatograph, Agilent automatic sampler, chromatographic columnNH2-50 4E (4.6 mm*250 mm), and differential detector Agilent G1362A; amobile phase is a 78% (v/v) acetonitrile-water mixed solution, a flowrate is 0.8 mL/min, and column temperature is set at 35° C.

Measuring Method of Galactooligosaccharide Yield:

Yield (%)=(Mass of the galactooligosaccharide in product/Mass of allsaccharides in product)×100%

The galactooligosaccharide yield is the sum of transferreddisaccharides, transferred trisaccharides and transferredtetrasaccharides.

EXAMPLE 1: PREPARATION OF SINGLE MUTANT AND WILD ENZYME

A β-galactosidase gene (SEQ ID NO.1) is connected to pMD19-T simple toobtain an AorE/pMD19-T simple plasmid, and the AorE/pMD19-T simpleplasmid used as a template is subjected to mutation by using Sac1m-F andSac1m-R as primers. An above PCR product is subjected to Dpn1 digestion,and the obtained plasmid is transformed into an escherichia colicompetent cell to obtain an AorE-M/pMD19-T simple plasmid.

Sac1m-F: TCCACAAGATCAGGGCTCTTGGTTTCAAC (mutant sites are underlined) Sac1m-R: GTTGAAACCA AGAGCCCTGA TCTTGTGGA (mutant sites are underlined) Preparation of wild enzyme: Snab1 and Not1 double-enzyme digestion isperformed on the AorE-M/pMD19-T simple plasmid and a pPIC9k vector,enzyme digestion products are connected by a T4 ligase, a connectionproduct is transformed into an Escherichia coli JM109 competent cell,and bacterium picking is performed to extract a plasmid, wherein theplasmid is named AorE-M/pPIC9k.

1. Preparation of recombinant bacterium pichia pastoris: theAorE-M/pPIC9k plasmid is electrically transformed into a KM71 yeastcompetent cell, wherein the recombinant bacterium is namedAorE-M/pPIC9k-KM71.

2. Preparation of single mutant: primers for N140C, F264W, F304Q andW806F mutations are respectively designed and synthesized, and theβ-galactosidase gene is subjected to site-directed mutagenesis.

PCR amplification of site-directed mutant coding gene: by using a rapidPCR technique, PCR is performed by using a vector AorE-M/pMD19-T simplecarrying a gene coding wild type maltooligosyl trehalose synthase as atemplate to respectively obtain mutated plasmids.

Mutagenic primers are as follows:

140m-F: CCGGTTCGTACATCTGTGCCGAGGTCTCA (mutant sites are underlined) 140m-R: TGAGACCTCGGCACAGATGTACGAACCGG (mutant sites are underlined) 806m-F: CTCGACGAGAATTTCACGGTCGGCGAGGA (mutant sites are underlined) 806m-R: TCCTCGCCGACCGTGAAATTCTCGTCGAG (mutant sites are underlined) 264m-F: AGCTATCCCCTCGGCTGGGATTGCGCAAACCCAT (mutant sites are underlined) 264m-R: ATGGGTTTGCGCAATCCCAGCCGAGGGGATAGCT (mutant sites are underlined) F304-Q: TCCAAGCGGGTGCTAATGACCCATGGGGTG (mutant sites are underlined) 264m-R: CACCCCATGGGTCATTAGCACCCGCTTGGA (mutant sites are underlined) 

Sequencing is respectively performed to confirm whether the coding geneof the β-galactosidase mutant is correct; Snab1 and Not1 double-enzymedigestion is performed on the plasmid carrying the mutant gene and thepPIC9k vector, enzyme digestion products are connected by a T4 ligase, aconnection product is transformed into an escherichia coli JM109competent cell, bacterium picking is performed to extract the plasmid,and the plasmid is transformed into a yeast KM71 cell to obtainrecombinant bacteria AorE-M/pPIC9k-N140C, AorE-M/pPIC9k-F264W,AorE-M/pPIC9k-F3040 and AorE-M/pPIC9k-W806F capable of expressing themutant gene.

EXAMPLE 2: CONSTRUCTION OF DOUBLE MUTANTS

By using the plasmid of the mutant N140C constructed by the Example 1 asa template for double mutations, according to the primers ofsite-directed mutagenesis designed by the Example 1, site-directedmutagenesis is performed on the plasmid carrying the gene coding themutant N140C by a rapid PCR technique to construct double mutantsN140C/F264W and N140C/W806F. Sequencing is respectively performed toconfirm whether a coding gene of the β-galactosidase double mutants iscorrect; Snab1 and Not1 double-enzyme digestion is performed on theplasmid with a correct sequencing result and the pPIC9k vector, enzymedigestion products are connected by a T4 ligase, a connection product istransformed into an escherichia coli JM109 competent cell, bacteriumpicking is performed to extract the correct plasmid, and the plasmid istransformed into a yeast KM71 cell to obtain recombinant bacteriaAorE-M/pPIC9k-N140C/F264W and AorE-M/pPIC9k-N140C/W806F capable ofexpressing the double mutant genes.

EXAMPLE 3: PREPARATION OF WILD BACTERIUM AND MUTANT β-GALACTOSIDASESOLUTIONS

The recombinant bacteria AorE-M/pPIC9k/KM71, AorE-M/pPIC9k-N140C,AorE-M/pPIC9k-F264W, AorE-M/pPIC9k-F304Q, AorE-M/pPIC9k-W806F,AorE-M/pPIC9k-N140C/F264W and AorE-M/pPIC9k-N140C/W806F are respectivelytransferred into the BMGY liquid culture medium, and are cultured at 30°C. for 24 h to obtain a bacteria solution. The bacteria solution iscentrifuged, all thalli are transferred into 50 mL of the BMMY liquidculture medium, and cultured at 30° C. for 5 days, wherein methanol ofwhich the final concentration is 0.75% is added every 24 h to performinduced enzyme production. A fermentation solution is centrifuged, andsupernate is a crude enzyme solution.

After the enzyme activity of the crude enzyme solution is measured, ONPGhydrolase activities of the wild type β-galactosidase (WT) and themutant are listed in Table 1.

TABLE 1 Enzyme activities of wild type β-galactosidase and mutantenzymes Enzyme Enzyme Activity (U/mL) WT 185.3 N140C 9.7 F264W 23.2F304Q 37.5 W806F 161.2 N140C/F264W 19.3 N140C/W806F 32.1

It can be seen from Table 1 that the mutants have lower oNPG hydrolaseactivity than the wild type. In this experiment, the enzyme activity andthe yield are not directly proportional, and the level of the hydrolaseactivity does not represent the level of the yield. The hydrolaseactivity is only used as a quantitative measure of the amount of enzymeadded.

EXAMPLE 4: OPTIMUM TEMPERATURE FOR PRODUCING GALACTOOLIGOSACCHARIDE BYTRANSFORMING LACTOSE BY USING MUTANT ENZYME

A 60% (W/V) lactose solution is prepared as a substrate, 60 g of lactoseis dissolved in 50 mL of pH4.5 50 mml/L acetic acid buffer, and thevolume of the solution is made up to 100 mL. Certain amounts of the wildenzyme and the double-mutant enzyme solution are added, so that thefinal enzyme activity of the enzyme solution is 2.5 U/mL; the enzymesolution is subjected to reaction at different temperatures, wherein thereaction for the wild type is performed for 10 h, the reaction for themutant is performed for 5 h, and the reaction time is determined by thepre-experiment; and the galactooligosaccharide yield is analyzed.

It can be seen from FIG. 1 that the optimum enzymatic transformationtemperature for the wild-type β-galactosidase is 60° C., the optimumenzymatic transformation temperature for the mutants is 40-60° C., andthe maximum yield of the wild enzyme is 35.7% while the maximum yield ofdifferent mutants is 47.7% to 59.8%, which is greatly improved ascompared with the wild enzyme. The maximum yield of the N140C/F264Wdouble-mutant reach 59.8%, which is about 1.67 times that of the wildenzyme. In addition, as can be seen from the accompanying drawing, thetemperature has little effect on the wild enzyme and the mutants at40-60° C., so the present invention is more suitable for industrializedapplication.

EXAMPLE 5: OPTIMUM pH FOR PRODUCING GALACTOOLIGOSACCHARIDE BYTRANSFORMING LACTOSE BY USING MUTANT ENZYME

A 60% (W/V) lactose solution is prepared as a substrate, 60 g of lactoseis dissolved in 50 mL of 50 mml/L acetic acid buffers with different pH,and each solution is made up to 100 mL. Certain amounts of the wildenzyme and the double-mutant enzyme solution are added, so that thefinal enzyme activity of the enzyme solution is 2.5 U/mL. The reactiontemperature of the wild type is 60° C., and the reaction temperature ofthe mutants is determined according to the optimum temperature in theExample 4, ranging from 40 to 60° C. The reaction for the wild type isperformed for 10 h, and the reaction for the mutants is performed for 5h, wherein the reaction time is determined by the pre-experiment; andthe galactooligosaccharide yield is analyzed.

It can be seen from FIG. 2 that except that the optimum pH of N140C is5, the optimum pH of other mutants is 4.5. The maximum yield of thewild-type enzyme reaches 35.7% while the maximum yield of differentmutants is 47.7%-59.8%, which is greatly improved as compared withcontrols. The maximum yield of N140C/F264W double-mutant reaches 59.8%,which is about 1.67 times that of the wild enzyme.

What is claimed is:
 1. A gene coding a mutant β-galactosidase enzymecomprising a variant of the amino acid sequence of SEQ ID NO:2 with oneor more mutations selected from N140C, F264W, F304Q, and W806F, whereinthe enzyme catalyzes conversion of lactose to galactooligosaccharide(GOS).
 2. A vector or recombinant cell carrying the gene of claim
 1. 3.A construction method of a recombinant bacterium expressing the gene ofclaim 1, comprising the following steps: (1) according to apredetermined mutant sites, designing mutagenic primers of site-directedmutagenesis, and performing site-directed mutagenesis by using a vectorcarrying a β-galactosidase gene as a template to obtain a gene encodinga β-galactosidase mutant; and constructing a mutant plasmid containingthe gene encoding the β-galactosidase mutant; (2) transforming themutant plasmid into a host cell; and (3) selecting a positive clone,performing a fermentation culture, and performing centrifuging to obtaina supernate from the fermentation culture, wherein the supernate is acrude enzyme solution of the β-galactosidase mutant.
 4. The methodaccording to claim 3, wherein the mutant plasmid is derived from a pETbasedor pPIC9k plasmid.